Single nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is altered. For example, a SNP might change the DNA sequence AAGGCTAA to ATGGCTAA. For a variation to be considered a SNP, it generally—but not always—occurs in at least 1% of the population. SNPs make up ˜90% of all human genetic variation, and occur every 100 to 300 bases along the 3-billion-base human genome. SNPs can occur in coding (gene) and noncoding regions of the genome. Many SNPs have no effect on cell function, but others have been associated with inherited traits, genetic diseases, age-associated diseases, and responses to drugs and environmental factors.
SNP genotyping assays are genetic tests that determine the presence of a sequence in nucleic acid, which can be used to detect the presence of SNPs —or other sequence variations in nucleic acids, e.g., microsattelites, deletions and insertions, duplications, and translocations. SNP genotyping assays can be separated into two groups based on mechanisms: (1) primer extension assays (including sequencing and PCR), and (2) hybridization assays (e.g., molecular beacons, microarrays, oligonucleotide ligation, and allele-specific endonuclease cleavage (e.g., invader assay)). Many assays require input of nucleic acid at quantities greater than typical yields of nucleic acid isolation procedures, and necessitate a nucleic acid amplification step to amplify nucleic acids in sufficient quantity to be detectable by the assay. The nucleic acid amplification steps use (1) sequence-specific primers to amplify regions of interest of nucleic acid (e.g. containing a SNP), and (2) high hybridization temperatures, necessary for sequence-specific primers to specifically hybridized and amplify template nucleic acid, which leads to specific amplification. In absence of high hybridization temperatures, the yield of amplified product drops and disappears from electrophoretic gels, and multiple background products appear (Henegariu, et. al., Biotechniques, 23:504 (1997)).
Nucleic acid amplification can be (1) thermocycling, e.g. PCR, using multiple heating cycles of 94° C., 55° C., and 72° C. for template denaturation, primer hybridization, and primer extension, respectively; or (2) isothermal, e.g., performed substantially at a single temperature, e.g. employing different mechanisms to bypass the denaturation step, and using single heating cycles of 37° C.-to-55° C. for primer hybridization and primer extension. Examples of isothermal amplification—capable of amplifying naturally occurring DNA or RNA—include Qβ replicase (Tyagi et al., PNAS USA 93:5395, 1996); self-sustained sequence replication, 3SR (Guatelli et al., PNAS USA 87:1874, 1990); strand displacement amplification, SDA (Walker et al., PNAS USA 89:392, 1992); and helicase dependent amplification (U.S. application Ser. No. 10/665,633). SNP genotyping assays requiring nucleic acid amplification—and its use of high hybridization temperatures—are not ideal for use in diagnostic assays, especially in kits and apparatuses in Point-of-Care (POC) settings and patient use. Towards fulfilling this need, a new combination of SNP genotyping assay and nucleic acid amplification was developed—whose nucleic acid amplification step can use heating cycles of less than 37° C., for example, room temperature.